Abstract
Previous work by our group described that human β-defensin-2 induces accumulation of extracellular adenosine (Ado) in E. coli cultures through a non-lytic mechanism causing severe plasmolysis. Here, we investigate the presence of AMP as a direct precursor and the involvement of a bacterial enzyme in the generation of extracellular Ado by treated bacteria. Following hBD-2 treatment, metabolites were quantified in the supernatants using targeted HPLC-MS/MS analysis. Microbial growth was monitored by optical density and cell viability was determined by colony forming units counts. Phosphatase activity was measured using chromogenic substrate pNPP. The results demonstrate that defensin-treated E. coli strain W releases AMP in the extracellular space, where it is converted to Ado by a bacterial soluble factor. An increase in phosphatase activity in the supernatant was observed after peptide treatment, similar to the effect of sucrose-induced osmotic stress, suggesting that the periplasmic 5'nucleotidase (5'-NT) is released following the plasmolysis event triggered by the peptide. Ado accumulation was enhanced in the presence of Co2+ ion and inhibited by EDTA, further supporting the involvement of a metallo-phosphatase such as 5’-NT in extracellular AMP conversion into Ado. The comparative analysis of hBD-induced Ado accumulation in different E. coli strains and in Pseudomonas aeruginosa revealed that the response is not correlated to the peptide's effect on cell viability, but indicates it might be dependent on the subcellular distribution of the nucleotidase. Taken together, these data shed light on a yet undescribed mechanism of host-microbial interaction: a human antimicrobial peptide inducing selective release of a bacterial enzyme (E. coli 5'-NT), leading to the formation of a potent immunomodulator metabolite (Ado).
Highlights
Multicellular organisms, including humans, are inhabited by a huge amount of microbes, collectively called microbiota
We had described the extracellular accumulation of Ado induced by defensin treatment in E. coli, as well as the presence of AMP as one of the purine-related metabolites released by treated bacteria
To gain some insight on the role of AMP as a precursor for the accumulated Ado, the levels of extracellular AMP accumulated by E. coli (DSM 1116) submitted to defensin treatment were measured by targeted LC-MS/MS at different time-points after the antimicrobial treatment
Summary
Multicellular organisms, including humans, are inhabited by a huge amount of microbes, collectively called microbiota. A complex balance involving commensal, mutualistic and infectious miccroorganisms must be kept to ensure harmonized co-existance: on the one hand several bacteria perform important functions, e.g. delivering nutrients including vitamins to the host; on the other hand, some can act as opportunistic pathogens. Host-microbial communication is a central piece in maintaining the homeostasis of abundant and diverse microbial communities such as the human intestinal microflora [2]. Disturbances in this delicate trade are known to have several clinical implications for the host as has been demonstrated for obesity [3], allergy [4] or inflammatory bowel disease (IBD) [5] and many more correlations are emerging [6]. A multitude of mechanisms are employed, both by the host and the microbiota, to control detrimental microbes and simultaneously foster beneficial ones [7]
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